Plant cell walls consist mainly of the large biopolymers cellulose, hemicellulose, lignin and pectin. Cellulose and hemicellulose constitute an important renewable and inexpensive carbon source for the production of fermentable sugars. Cellulose consists of D-glucose units linked together in linear chains via beta-1,4 glycosidic bonds. Hemicellulose consists primarily of a linear xylan backbone comprising D-xylose units linked together via beta-1,4 glycosidic bonds and numerous side chains linked to the xylose units via beta-1,2 or beta-1,3 glycosidic or ester bonds (e.g., L-arabinose, acetic acid, ferulic acid, etc).
Filamentous fungi of the phylum (division) Ascomycota, including various Penicillium, Phanerochaete, Agaricus, Neurospora, Humicola, Fusarium, Chaetomium, Magnaporthe, Aspergillus and Trichoderma species, have a key role in degradation of the most abundant polymers found in nature, cellulose and hemicellulose. Trichoderma reesei (the asexual anamorph of Hypocrea jecorina) is an important industrial source of cellulase and hemicellulase enzymes. The term cellulase (or cellulase enzymes) broadly refers to enzymes that catalyze the hydrolysis of the beta-1,4-glucosidic bonds joining individual glucose units in the cellulose polymer. The catalytic mechanism involves the synergistic actions of endoglucanases (E.C. 3.2.1.4), cellobiohydrolases (E.C. 3.2.1.91) and beta-glucosidase (E.C. 3.2.1.21). The term hemicellulase broadly refers to enzymes that catalyze the hydrolysis of the various glycosidic bonds joining individual xylose, arabinose, mannose, galactose and other moieties in the hemicellulose polymer. Hemicellulases include, for example, endo-1,4-beta-xylanases (EC 3.2.1.8), beta-mannanases (EC 3.2.1.28), alpha-L-arabinofuranosidases (EC 3.2.1.55), 1,4-beta-xylosidase (EC 3.2.1.27) and alpha-glucuronidase (EC 3.2.1.139).
Trichoderma reesei is a commonly used industrial species of filamentous fungi for the production of biomass degrading enzymes such as cellulases and hemicellulases. Analysis of the secretome of T. reesei strain RutC30 revealed the presence of 31 secreted glycosyl hydrolases when grown in media supplemented with pretreated corn stover (Nagendran et al., 2009) Studies of the secretome of F. graminearum grown on hop cell wall identified that at least 45% of the secreted proteins are involved in plant cell wall degradation, with 25, 19 and 11 different proteins for hemicellulose, pectin and cellulose degradation, respectively (Phalip et al., 2005).
Sequencing and analysis of the T. reesei genome has revealed the presence of 10 genes encoding cellulase and 16 genes encoding hemicellulases (Martinez et al., 2008). These include two cellobiohydrolases, eight endoglucanases, four xylanases, two alpha-L-arabinofuranosidases, and a beta-mannanase. T. reesei also produces a number of accessory enzymes that assist in the generation of monosaccharides from the cellulose and hemicellulose, including acetyl xylan esterase, beta-xylosidase and several beta-glucosidases (de Vries and Visser, 2001; Aro et al., 2005, and references therein). However, when compared with the genomes of other filamentous fungi, the T. reesei genome has surprisingly few genes encoding glycoside hydrolases (total 200) (Martinez et al., 2008). For example, Aspergillus oryzae, Aspergillus fumigatus, Aspergillus nidulans and Fusarium graminearum encodes 285, 263, 247 and 243 glycosyl hydrolases, respectively (Martinez et al., 2008).
The production of plant cell wall degrading enzymes such as cellulases, hemicellulases, ligninases and pectinases, by filamentous fungi is regulated mainly at the transcriptional level in response to available carbon sources. Glucose represses cellulase gene expression through the action of transcriptional regulators such as cre1 (Strauss et al., 1995,). Under glucose-limiting conditions, cellulase transcription is derepressed, with full activation of transcription requiring the presence of a cellulase-inducing carbohydrate, or inducer, such as cellulose, or beta-linked disaccharides such as cellobiose, sophorose, gentiobiose and lactose (Ilmen et al., 1997), while activation of hemicellulase transcription is dependent on the presence of xylan or its derivatives (xylose, xylobiose, arabinose) in the growth media (Margolles-Clark et al., 1997).
The transcriptional regulator XlnR (xylanase regulator), initially identified in Aspergillus niger, controls the transcription of about 20-30 genes encoding hemicellulases and cellulases (Stricker et al, 2008 and references therein). Moreover, the extracellular xylan degradation and intracellular D-xylose metabolism is coupled via the transcriptional regulation of the xyrA (D-xylose reductase-encoding) gene by XlnR (Hasper et al, 2000). The orthologous transcription factors in T. reesei, Xyr1 (xylanase regulator 1) and Aspergillus oryzae (Ao XlnR) are also a general regulators of cellulase and hemicellulase gene expression (Striker et al, 2006; Marui et al, 2002). Studies of several other identified regulators of xylanase expression in fungi are limited to the regulation of hemicellulase genes (Tamayo et al, 2008; Rao et al, 2002; Calero-Nieto et al, 2007). For examples, it has been shown that deletion of an orthologous transcription factor to Xyr1 from Fusarium graminearum did not affect the basic expression levels of xylanases and cellulases but did prevent high inducible expression (Brunner et al, 2007). This finding is in contradiction to the studies with Trichoderma and Aspergillus, where the knock out of the corresponding regulator abolishes cellulase and xylanase expression completely. These observations led to a system for production of homologous and/or heterologous proteins using XlnR regulated promoter along with overexpression of xylanase regulator, XlnR, from multiple gene copies (U.S. Pat. No. 6,177,261 B1, 2001).
Xylanase regulators, such as Xyr1 from Trichoderma and XlnR from Aspergillus, belong to class III zinc binuclear cluster protein family found exclusively in fungi and possess a conserved amino acid motif (CX2CX6CX5-12CX2CX6-8C) at the N-terminal part of the protein (MacPherson et al., 2006). This class of transcription factors is unique in containing only one zinc finger that binds two zinc atoms. Xylanase regulators bind 5′-GGC(T/A)3-3′ response elements in the promoters of target genes, and may interact with DNA as monomers, homodimers or heterodimers (MacPherson et al., 2006; Stricker et al., 2008). Several studies have shown that T. reesei Xyr1 is essential for the expression of all major (hemi)cellulase genes (Stricker et al., 2006) and that it binds to xylanase 1, 2 and 3 gene promoters (Rauscher et al, 2006; Stricker et al, 2007; Furukawa et al, 2009). However, in vitro binding of T. reesei Xyr1 to cellulase gene promoters was only recently demonstrated (Furukawa et al, 2009; Ling et al., 2009). In silico analysis has revealed that the 5′-GGC(T/A)3-3′ motifs are widespread as single sites in 5′-upstream region of all Xyr1-regulated genes in T. reesei (Furukawa et al, 2009). However in vitro studies of Xyr1 binding to selected motifs revealed that only several of them can be recognized by this transcription factor (Furukawa et al, 2009).
Other functional domains have been identified for A. niger XlnR by loss-of-function mutations and rational design mutagenesis analyses (Hasper et al., 2004). These studies demonstrated that the second putative coiled-coil domain is involved in the nuclear localization of the protein. Protein structure predictions suggest the presence of two coiled-coil domains at similar positions in A. niger XlnR and T. reesei Xyr1. Thus, the second coiled-coil domain of T. reesei Xyr1 may likewise be responsible for its transport into the nucleus. The C-terminus of XlnR is essential for transcriptional regulation; deletion of 78 C-terminal amino acids causes increased expression of XlnR target genes, even under glucose repression conditions, suggesting this region dampens transcriptional activation by XlnR (Hasper et al., 2004). However, certain single-amino acid mutations in this region such as Tyr864Phe, Leu823Ser and Tyr864Asp lead to severely diminished activation by XlnR (Hasper et al., 2004).
Although A. niger XlnR and T. reesei Xyr1 share similarities in structure and in consensus binding sites, there is evidence to suggest that these factors interact with promoters via different mechanisms. For example, it was suggested that A. niger XlnR binds as a monomer (Hasper et al., 2004), while T. reesei Xyr1 binds to an inverted repeat within a regulated gene promoter, as either a homo- or a heterodimer with Ace2, a known positive regulator of cellulase expression in T. reesei (Stricker et al., 2006, 2008). It is also hypothesized that regulation of hemicellulase and cellulase gene expression in T. reesei by Xyr1 and Ace2 may involve phosphorylation and recruitment of other regulatory proteins (Stricker et al., 2008). T. reesei Xyr1 also has an antagonistic relationship with Ace1, a negative regulator of cellulase genes, through a possible competition of the two factors for the same binding site within cellulase promoters (Stricker et al, 2006). Putative Ace1-encoding genes were isolated from several other fungal species, such as Aspergillus nidulans, Talaromyces emersonii, and Neurospora crassa (Aro et al, 2005); however, their possible interaction with XlnR and their participation in transcriptional activation of hydrolase-encoding genes has not yet been shown (Stricker et al., 2006).
T. reesei produces low levels of xylanase activity under cellulase-inducing conditions; however, the enzyme system produced by cultures of T. reesei growing on xylan, xylose and arabinose, is enriched in hemicellulase activities relative to cellulase activities (Mach and Zeilinger 2003; Margolles-Clark et al., 1997; Xiong et al., 2004). This could be beneficial when the goal is to produce an enzyme composition having high xylanolytic activity relative to cellulase activity, as in the animal feed and pulp and paper industry. U.S. Pat. Nos. 6,300,112 and 5,298,405 disclose the use of cellulase-deletion strains as an alternative approach to the production of hemicellulase-enriched enzyme preparations for use in animal feed and for bio-bleaching applications
There are situations in which it is desirable to produce cellulase mixtures with a high cellulase specific activity from fungal cultures using carbohydrate sources comprising mainly xylose and other pentose sugars derived from hemicellulose, such as those produced by chemical treatments of lignocellulosic biomass. These may contain HDC or CIC However, such carbon sources result in enzyme compositions containing high hemicellulase activity with decreased cellulase specific activity, and, as a consequence, higher dosages of total protein are needed for effective hydrolysis of cellulose. Further, the production and secretion of hemicellulase enzymes uses cell energetic and secretion pathway resources and limits the cellulase expression and secretion capacity of the host cell.
It has been reported that a combination of xylan-derived carbohydrates with cellulase inducers such as cellobiose or lactose can lead to different proportions of cellulase and hemicellulase in the protein mixture secreted by Trichoderma reesei (Zeilinger, S., et al., 1996,). In addition, it has been found that concentrations of inducer (need to define) of 8 (check)-15% can improve protein production on hemicellulose derived carbohydrate (HDC) almost up to the levels produced when cellulase inducing carbohydrates are used as the carbon source. (See co-pending U.S. application Ser. No. 12/200,492). However, due to high cost of inducing carbohydrates, the use of such mixtures on a large scale can significantly increase enzyme production costs. Moreover, a significant proportion of such an enzyme mix will still be composed of hemicellulases. Consequently, due to the high content of hemicellulases, and the requirement of adding cellulase inducing carbohydrates, the production of cellulase on hemicellulase derived carbohydrates is currently not cost effective.
Thus, there is a need in the art for a cost-effective method of producing a cellulase mixtures containing low levels of hemicellulase activity from filamentous fungi using primarily hemicellulose derived carbohydrate (HDC) in the absence of the cellulase inducing carbohydrates, such as cellulose, or β-linked disaccharides such as cellobiose, sophorose, gentiobiose and lactose, or containing low levels of such carbohydrates.